PROJECT SUMMARY/ABSTRACT The focus of this proposal is to understand the fundamental structure and function of replisomes responsible for DNA replication and the responses to a damaged DNA template to further treatments for a variety of disease states, from viral infection to cancer, and for discovery of new potential therapeutic targets. The T4 bacteriophage replication system serves as an important model system because all the functions of the more complex human replisome are preserved in a smaller ensemble of T4 proteins. An extensive body of literature exists on the functioning of the individual T4 replication proteins. Aim 1a of this proposal is to understand the coordination of leading- and lagging-strand DNA synthesis by measuring the distribution of Okazaki fragment sizes and gap lengths using Single Molecule, Real-Time (SMRT) Sequencing. Aim 1b is to determine the solution orientation and dynamics exhibited by the two holoenzymes within a replisome using single-molecule FRET and fluorescence polarization measurements. Aim 1c strives to unify complex function with structure by solving the structures of the T4 replisome subassemblies followed by the complete T4 replisome using cryo- EM. The collective findings will provide a detailed and comprehensive picture of DNA replication that can also provide insight on the human replication system. DNA damage tolerance (DDT) pathways, including translesion synthesis (TLS), allow the bypass of DNA damage postponing its repair and allowing DNA replication to continue in order to complete the cell cycle. To combat the onslaught of diverse DNA lesions, a complex process involving TLS polymerases, a variety of auxilary proteins, and post-translational modifications (PTMs) participate in the human DDT process. In Aim 2a, in situ biotinylation utilizing chimeric APEX2 constructs in living cells and subsequent proteomic mapping will be used to identify proximal proteins directly involved in the bypass of lesions caused by various DNA damaging agents, including UV radiation, chemotherapy by cisplatin, and exposure to benzo[a]pyrene. In Aim 2b, the same in situ biotinylation reactions will be expanded to elucidate the temporal evolution of proteins participating in DDT from lesion bypass to gap filling. Various times (established by observing DNA damage foci) corresponding to translesion synthesis (insertion stage), polymerase switching (extension stage), and replication (gap filling stage) will be pursued. The goal of Aim 2 is to map the events and associated protein participants that define the DDT process as a function of time, thus providing a mechanistic basis for in vivo DDT.